Emerging research in cancer therapy is focused on exploiting the biochemical differences between cancer cell and normal cell metabolism. The Warburg effect is a fundamental change in many malignant cancer cells and is the shift in energy metabolism from oxidative phosphorylation to aerobic glycolysis. The common perception is that this metabolic reprogramming provides the cellular energy required for unregulated cell growth, invasion, and metastasis. Human pancreatic ductal adenocarcinoma (PDAC) is an incurable and highly aggressive human cancer. The median survival of the 75-80% of patients with malignant PDAC at the time of initial diagnosis is 6-months. Standard chemotherapy with the cytolytic drug gemcitabine provides a slight survival benefit. Thus, there is an unmistakable and critical unmet need for new therapies to treat patients with pancreatic cancer. The overall goal of this project is to develop new therapeutic approaches to inhibit PDAC malignancy. The significance of the proposed work lies in the use of relatively nontoxic mitochondria-targeted cationic drugs in combination with glycolytic and glutaminolytic energy metabolism inhibitors to decrease pancreatic cancer cell proliferation and metastasis. The overarching hypothesis is that a combination of glycolytic, glutaminolytic, and/or mitochondrial metabolism inhibitors with standard therapies will deplete ATP, decrease energy sensing, proliferation, and migration in vitro, and inhibit aerobic glycolysis and human PDAC tumor growth and metastasis in vivo. Studies in Aim 1 will use innovative high-throughput and mass spectroscopy-based metabolomics approach to investigate bioenergetic changes in glycolysis, tricarboxylic acid cycle, and glutaminolysis in human primary PDAC cells treated with mitochondria-targeted cationic agents, and/or inhibitors of energy metabolism. Aim 2 will use cell culture approaches to define the role for bioenergetic metabolism inhibitors in activating energy regulatory signaling pathways and altering PDAC growth, invasion, and migration. Aim 3 will use preclinical hyperpolarized magnetic resonance and bioluminescence imaging techniques to screen the in vivo efficacy of targeted drugs that inhibit energy metabolism, alone or in combination with traditional chemotherapy to mitigate PDAC growth and metastasis. The overall impact of the proposed work is two-fold: First, it will advance our understanding of the role of metabolism, energetics, and energy sensing in pancreatic cancer malignancy and second it will engender the design and testing of drugs that stifle energy production and which may ultimately be translated to the clinic.